The goal of this project is to contribute towards a comprehensive understanding of the biochemical and genetic parameters governing tRNA function and codon recognition in the genetic code. Employing protein synthesis in vitro in E. coli extracts directed by sequenced natural mRNA, or by sequenced DNA in a coupled transcription-translation system, we will be able to determine the specific coding properties of pure tRNAs by their ability to transfer their cognate amino acids into known positions in the protein, encoded by known codons. Peptide mapping techniques will be used to isolate specific portions of the protein. Using this method as our assay system of tRNA function, we will examine the following questions: 1) Codon recognition by E. coli tRNA Ser and tRNA Leu families of tRNAs under "normal" conditions; 2) the effects of ribosomal mutations on tRNA coding properties, including mutations in ribosomal "flexibility," and "relaxed" mutants; 3) the analysis of misreading phenomena under conditions when a specific aminocylated tRNA is absent, either due to a tRNA-synthetase mutation or due to amino acid "starvation" in vitro; 4) codon recognition by mutant and undermodified tRNAs, obtained from cells with mutations in tRNA modification enzymes, or from relaxed auxotrophic cells subjected to nutritional manipulations (leading to the production of undermodified tRNAs); and 5) examination of effects of rate-limiting elongation upon protein synthesis, either by substrate limitation in tRNA-synthetase deficient extracts, or by tRNA mutations leading to partial growth defects of phage. The above experiments will help lay the groundwork for a revision of the wobble hypothesis, taking into account the already apparent greater flexibility of tRNA-mRNa interactions as well as the role of the ribosome. The concept of a "translational hierarchy" of tRNAs potentially capable of responding to a given codon will be explored. Further insight into the role of tRNA in regulation may be gained by studying protein synthesis under rate-limiting elongation. Thus, we will obtain information on codon recognition, fidelity, and efficiency of translation processes, under normal wild type conditions as well as under conditions when the protein synthesis apparatus in perturbed by mutations.